To simplify a quantitative immobilization procedure of ligands with maintaining their activities, we developed an automated preparation method using an alginate hydrogel partially formed in a capillary. After a sodium alginate solution containing a ligand was injected into the capillary, a background solution containing Ca(2+) was then introduced into the sodium alginate solution zone by applying an appropriate voltage for the hydrogelation, resulting in encapsulation of the ligand by the formed alginate hydrogel. According to the estimated binding capacity for biotin by the encapsulated avidin, the injected avidin was immobilized quantitatively by the formed hydrogel with keeping its affinity. When avidin (3.5-35.2 ng) was immobilized by the proposed method, the immobilization efficiency was estimated to be almost 100%. Furthermore, by using the prepared capillary, biotinylated fluorescein was specifically trapped and separated due to the affinity of the encapsulated avidin. By the change of pH of the background solution, the concentrated analytes could be easily eluted, resulting in 90% recovery with high reproducibility by using 1.18 fmol biotinylated sample.